A cell always requires the uptake of an amino acid as a nutrition, and such a function is exerted by an amino acid transporter which is a membrane protein existing in a cell membrane. The amino acid transporter is distributed in a specific site in each tissue in a multicellular organism and plays an important role in expressing the specific function of each tissue. For example, in kidney cells and small intestine, it plays a role for epithelial absorption of amino acid in lumen and, in nerve tissues, it is in charge of recovery of amino acid as a neurotransmitter released as a result of neurotransmission and also of supply of amino acid as a precursor for neurotransmitter to nerve cells. Further, it exists in blood-brain barrier and placental barrier and makes permeation of the amino acid possible.
With regard to an amino acid transport mechanism, its identification and classification have been conducted using cultured cells and membrane specimens since 1960's and, reflecting the multiplicity of amino acid molecules, many transport systems have been described. However, there has been no independent transport system for each amino acid but most of the amino acid transports have been conducted by a few kinds of transport systems which transport several amino acids having similar side chains (Christensen, Physiol. Rev., volume 70, page 43, 1990).
Transport of acidic amino acids such as glutamic acid and aspartic acid having carboxyl group on a side chain has been believed to be carried out by both of a sodium-dependent transporter which requires sodium ion for its function and a sodium-independent transporter which does not require sodium ion for its function.
However, in a conventional method, it is difficult to analyze the details of the transport of an amino acid or its analogue via the acidic amino acid transport system and the in vivo functional roles, and it has been desired to enable a detailed functional analysis by isolating a gene of acidic amino acid transporter responsible for the function of the acidic amino acid transport system.
With regard to sodium-dependent acidic amino acid transporters, five kinds of glutamate transporters—EAAC1, GLT-1, GLAST, EAAT4 and EAAT5—have been cloned (Kanai, Curr. Opin. Cell Biol., volume 9, page 565, 1997; Kanai and Endou, Curr. Drug Metab., volume 2, page 339, 2001).
With regard to sodium-independent transporters, LAT1 (Kanai, et al., J. Biol. Chem., volume 273, pages 23629-23632, 1998) and LAT 2 (Segawa, et al., J. Biol. Chem., volume 274, pages 19745-19751, 1999) have been cloned as neutral amino acid transporters corresponding to a transport system L. It was also shown that LAT1 and LAT2 function only when they coexist with a cofactor 4F2hc which is a single membrane-spanning type protein. LAT1 shows an exchange transport activity which transports large-sized neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine and histidine while LAT2 shows a broad substrate selectivity transporting small-sized neutral amino acids such as glycine, alanine, serine, cysteine and threonine in addition to large-sized neutral amino acids and they are not acidic amino transporters.
With regard to proteins analogous to LAT1 and LAT2, the above-mentioned y+LAT1 and y+LAT2 having the functions of a transport system y+L which transports neutral amino acids and basic amino acids have been cloned (Torrents, et al., J. Biol. Chem., volume 273, pages 32437-32445, 1998). It was also revealed that both of y+LAT1 and y+LAT2 function only when being coexisting with a cofactor 4F2hc. y+LAT1 and y+LAT2 mainly transport glutamine, leucine and isoleucine as neutral amino acids and do not transport acidic amino acids.
With regard to a transporter which requires a cofactor 4F2hc for expressing its function, Asc-1 which is a protein analogous to LAT1 to LAT2 was cloned (Fukasawa, et al., J. Biol. Chem., 275: 9690-9698, 2000). Asc-1 selectively transports alanine, serine, cysteine, threonine, glycine, etc., shows a substrate selectivity of amino acid transport system asc and does not transport acidic amino acids.
With regard to a transporter which requires another cofactor rBAT having an analogous structure to 4F2hc for expressing its function, BAT1 which is a protein analogous to LAT1 and LAT2 was cloned (Chairoungdua, et al., J. Biol. Chem., 274: 28845-28848, 1999). BAT1 transports cystine, neutral amino acids and basic amino acids and does not transport acidic amino acids.
As described above, molecular entity of a transporter which functions by binding to 4F2hc and rBAT was characterized and, the presence of a group of transporters which achieves a transport ability by forming heterodimer with a single membrane-spanning type protein and a heterodimeric amino acid transporter family was established.
Further, with regard to a transporter requiring a cofactor 4F2hc for expressing its function, xCT which is a protein analogous to LAT1 and LAT2 was cloned (Sato, et al., J. Biol. Chem., 274; 11455-11458, 1999). xCT transports cystine, glutamic acid and sodium aminoadipate in a sodium-independent manner and corresponds to an amino acid transport system Xc. xCT needs a negative charge of side chain of amino acid for recognition of substrate and is classified under sodium-independent acidic amino acid transporters (Kanai and Endou, Curr. Drug Metab., volume 2, page 339, 2001).
xCT transports glutamic acid but does not transport aspartic acid and its transport is suppressed by cystine. In addition, xCT is a transporter where expression is induced by oxidative stress and, except a few cases, its expression in common normal tissues is not detected. However, it has been reported that there is a sodium-independent glutamic acid and aspartic acid transporter which is not suppressed by cystine (Christensen, Physiol. Rev., volume 70, page 43, 1990) and it has been suggested that there is a sodium-independent acidic amino acid transporter other than xCT which has not been identified.
Further, Asc-2 which is a protein having an analogous structure to LAT1 and LAT2 and binds to unidentified protein other than rBAT or 4F2hc was cloned (Chairoungdua, et al., J. Biol. Chem., 276: 49390-49399, 2001). Asc-2 is not expressed in a cell membrane by itself, however, by preparing a fusion protein with 4F2hc or rBAT, it transfers to a cell membrane as a fusion protein and a transport activity can be detected. When Asc-2 is expressed in a cell membrane as a fusion protein with 4F2hc or rBAT, it shows a characteristic of a sodium-independent neutral amino acid transport system asc.